The polypeptide compounds consist of an assembly of homologous or heterologous amino acid units onto which molecules are grafted whose biological and/or pharmacological activity is preserved.
It may involve, for example, polylysine compounds. These compounds have been described in particular in the patent FR9413861.
The polypeptide compounds such as polylysine can be used for numerous biological, food-processing, medical, pharmaceutical, cosmetic and environmental applications.
So that these compounds are distributed as raw materials for these applications, in particular as raw materials for pharmaceutical usage (RMPU), it is necessary to be able to issue reliable analytical reports making possible their identification and their characterization. Today, however, there is no specific method that makes it possible to identify and to characterize in a reliable, pertinent, and rigorous way the polypeptide compounds that are substituted in general and polylysine compounds in particular. Analytical proposals have been carried out for these compounds, but none are satisfactory.
It is possible to cite, for example, the study carried out in 2002 by the Waters Company (St. Quentin en Yvelines (78)), which describes in particular the identification of components grafted onto polylysine, by coupling liquid chromatography and positive and negative electrospray mass spectrometry, but this method does not make it possible to separate the analytes correctly. Likewise, a study carried out in 2003 by NMR (nuclear magnetic resonance) DOSY (“Diffusion Ordered Spectroscopy”), the NMRtec laboratory of the Montpelier Pharmaceutical Department (34), according to the NMR DOSY analytical report of GEMSEP01b, reference 20030739, describes a method that makes it possible to detect the presence of polylysine compounds that are expected in the mixture, to identify the skeleton of the polylysine, but this method does not make it possible to identify in a reliable way the different molecules that are grafted with low concentrations of the preparation.
The use of the electrospray liquid chromatography technique coupled to a mass spectrometer (HPLC/ESI/QTOF-MS) was reevaluated and also described in the report of the Université René DESCARTES, Paris V, Pharmaceutical Department (75) in 2003-2004. There again, this method does not make it possible to quantify the grafted molecules of the polylysine compounds.
The verification of the grafting of haptenes onto polylysine was also carried out by a MALDI-TOF and MALDI/MS mass spectrometer in the laboratory of the Institute of Molecular Sciences of the Université de Bordeaux (33) in 2005 and 2008.
This technique confirms the presence of small grafted molecules but did not allow them to be identified and characterized.
The method by steric exclusion chromatography (SEC) has also been tested according to the results of the Université de Bordeaux, but it did not provide any significant results relative to polylysine and its compounds.
It is also possible to cite the implementation of immuno-enzymatic metering by Elisa test of polylysine compounds grafted with amino acids according to a report from the Gemac Company for the clinical test of 2003, No. 2002SEP01. Although this method made it possible to calculate the curve of the metering of the compound that is grafted onto polylysine, it did not make it possible to identify and meter the grafted molecules/polylysine ratio correctly. In addition, this method does not demonstrate possible molecular artifacts.
Other techniques have been tested, but none of these methods is reliable and can be applied to all of the polypeptide compounds grafted with various molecules.